Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of miRNA-Bound Ago2 to RNA Processing Bodiesin vitro

Phase separation to insoluble membrane-less organelles is a major way of activity regulation of specific proteins in eukaryotic cells. miRNA-repressed mRNAs and Ago proteins are known to be localized to RNA-processing bodies, the subcellular structures which are formed due to assembly of several RNA binding and regulatory proteins in eukaryotic cells. Ago2 is the most important miRNA binding protein that by forming complex with miRNA binds to mRNAs having cognate miRNA binding sites and represses protein synthesis in mammalian cells. Factors which control compartmentalization of Ago2 and miRNA-repressed mRNAs to RNA processing bodies are largely unknown. We have adopted a detergent permeabilized cell-based assay system to follow the phase separation of exogenously added Ago2 to RNA processing bodies in vitro . The Ago2 phase separation process is ATP dependent and is influenced by osmolarity and salt concentration of the reaction buffer. miRNA binding of Ago2 is essential for its targeting to RNA processing bodies and the compartmentalization process gets retarded by miRNA binding “sponge” protein HuR. This assay system found to be useful in identification of amyloid beta oligomers as miRNA-activity modulators which repress miRNA activity by enhancing Ago2-miRNP targeting to RNA processing bodies. Graphical Abstract miRNA bound Ago2 gets phase separated in vitro to RNA processing bodies (PBs) in detergent permeabilized mammalian cells. Phase separation of Ago2 to PBs is controlled by presence of ATP and RNA. Amyloid beta oligomers retard dynamics of Ago2 bodies to inhibit miRNA function and enhance PB targeting of Ago2 miRNPs. microRNA binding protein HuR can rescue Ago2 miRNP from PBs and inverse the effect of amyloid beta oligomers.